Viral epitopes that are recognized by both HLA class I-restricted (CD8+) and class II-restricted (CD4+) CTL have been defined for a type A influenza virus nucleoprotein (NP) peptide encompassing residues 335 to 349 of NP that are restricted by HLA-B37 and HLA-DQw5, respectively. Amino acid residues that are critical for recognition of this NP determinant in the context of HLA-B37 and HLA-DQw5 were investigated by the use of panels of truncated and alanine-substituted NP peptides. The results showed that the class I- and class II- restricted CTL lines recognize similar but distinct epitopes, and different structural features of the NP peptides are required for presentation by HLA-B37 and HLA-DQw5. Comparison of the amino acid sequences of the NP peptide presented by HLA-B37 and HLA-DQw5 with other peptides known to be presented by both class I and class II molecules revealed a common motif among these peptides. Multiple sclerosis (MS), a human demyelinating disease, is thought to be caused by an autoimmunogenic process, and myelin basic protein (MBP) is considered a likely autoantigen. Studies of T cell lines (TCL) responding to different parts of the MBP molecule have indicated that amino acids 87 through 106 contain an immunodominant epitope of MBP. The fine specificity of 29 CD4+ cytotoxic, long term, and limiting dilution TCL that had been generated against whole MBP and were derived from four MS patients and two healthy relatives was dissected using truncated and alanine-substituted peptides for the 87-106 peptide, and the TCR alpha and beta chain usage of 15 CD4+ TCL was determined. The results showed that the CD4+ cytotoxic T cell response to the immunodominant MBP peptide 87-106 demonstrates a high degree of heterogeneity in fine specificity and TCR usage. These findings indicate that specific immunotherapies aimed at TCR in MS will probably be more complicated than previously anticipated. To determine whether similar or dissimilar molecular features of class I molecules are involved in the presentation of structurally distinct peptides, we used site-directed mutagenesis to investigate the influence of different peptide-binding pockets of the HLA-A2.1 molecule on the presentation of three different viral peptides. HTLV-I Tax peptide 12- 19, HCMV gB 619-628, and influenza M1 58-66 are minimal peptides that induce HLA-A2.1-restricted noncrossreactive CTL. These results indicated that common structural features in HLA-A2 determine the binding of different peptides, and help to provide a plausible explanation for how structurally diverse peptides bind to HLA-A2.